Revealing the glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1, serotype Ogawa with the BSA protein carrier using LC‐ESI‐QqTOF‐MS/MS

Abstract

In this manuscript, we present the determination of glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1 serotype Ogawa to BSA using nano- liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectroscopy (LC-ESI-QqTOF-MS/MS). The matrix-assisted laser desorption/ionization-TOF/TOF-MS/MS analyses of the tryptic digests of the glycoconjugates having a hapten:BSA ratio of 4.3:1, 6.6:1 and 13.2:1 revealed only three glycation sites, on the following lysine residues: Lys 235, Lys 437 and Lys 455. Digestion of the neoglycoconjugates with the proteases trypsin and GluC V8 gave complementary structural information and was shown to maximize the number of recognized glycation sites. Here, we report identification of 20, 27 and 33 glycation sites using LC-ESI-QqTOF-MS/MS analysis of a series of synthetic neoglycoconjugates with a hapten:BSA ratio of, respectively, 4.3:1, 6.6:1 and 13.2:1. We also tentatively propose that all the glycated lysine residues are located mainly near the outer surface of the protein.

Mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of Vibrio cholerae, serotype Ogawa and the recombinant tetanus toxin C‐fragment carrier

“…[20–23] Based on this, the m/z value of the peptides that did not match to any protein were compared with a table model containing hypothetical molecular weight values of all possible hapten hexasaccharide conjugated to lysine residues of tryptic peptide of the tetanus toxin protein. Subsequently, the mass spectra of each possible glycated peptide were manually processed, looking for the carbohydrate-linker signature product ions, as well as the corresponding peptide sequence.…”

“…As observed in our previous studies, all the identified glycation sites are located exclusively on lysine residues. [20–23] This suggests that while the squaric acid chemistry allows an amino acid specific conjugation, it does not allow a site specific conjugation.…”

“…[12,20–22] Recently, we reported on the determination of the glycation sites of several different vaccine models prepared by dialkyl squarate chemistry: a synthetic tetrasaccharide hapten of Bacillus anthracis –BSA conjugate vaccine, a synthetic lactose-BSA conjugate and neoglycoconjugates from the terminal monosaccharide hapten of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa and BSA. [20–23] Our strategy then, as well as during the present work, was based on the enzymatic digestion of the hapten–BSA glycoconjugates, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the digests.…”

Direct targeted glycation of the free sulfhydryl group of cysteine residue (Cys‐34) of BSA. Mapping of the glycation sites of the anti‐tumor Thomsen–Friedenreich neoglycoconjugate vaccine prepared by Michael addition reaction

“…For the determination of the average number of carbohydrate‐spacer moieties linked to BSA (TF antigen:BSA), the two glycoconjugates were analyzed by matrix assisted laser desorption/ionization‐mass spectrometry (Fig. a and b and Table ) . The MALDI‐TOF‐MS was characterized by the formation of a protonated molecular ions [M + H] + at m/z 67 599 and 70 905 and their TF–BSA ratios were calculated to be individually 2:1 and 8:1.…”

“…BSA is one of the most widely studied proteins used as a carrier protein for the synthesis of neoglycoconjugates. [27,[37][38][39][40][41] BSA contains 35 cysteine residues linked by S-S disulphide bonds among which, however, there is only a single buried free cysteine residue (Cys34) [42] that has been systematically used for other conjugation. [43,44] It is known that the 35 cysteine residues of BSA form 17 disulphide bonds at these positions (1) (17) 581-590 .…”